Dendritic cells are involved in the initiation of both innate and adaptive immune responses. Direct contact with many pathogens leads to the maturation of DCs, which is characterized by an increase in antigen presentation, expression of costimulatory molecules and subsequent stimulation of naive T cells in lymphoid organs. We found immature DCs to be activated and to differentiate into mature DCs in response to Aspergillus antigens. In contrast, we showed CMV infection with certain (endotheliotropic) strains to down-regulate the expression of MHC class I and II and of co-stimulatory molecules as well as to inhibit DC maturation. Here we used oligonucleotide microarrays to test to what extent monocyte-derived immature DCs respond to Aspergillus fumigatus and its cell wall components and to HCMV variants. Immature dendritic cells (purity > 80%) were generated from purified peripheral blood monocytes under serum free conditions. DCs were then cultured with Aspergillus hyphae, a protein precipitate of Aspergillus fumigatus supernatant (PpSAB), human cytomegalovirus strains AD169 and TB40E as well as Pamp3cys, which recognizes and interacts with TLR2 and TLR4. As a positive control lipopolysaccharide and as negative control mock-infected and unstimulated DCs were used. After incubation for 8 h, total RNA was extracted, followed by cDNA synthesis, in vitro transcription using biotinylated NTP and hybridization to microarrays for 16 h at 45Â°C. To control for DC maturation and stimulation, TNF- and IL12p40 expression was documented by real-time RT-PCR. Stimulation experiments with each pathogen and component were repeated from four independent donors. Expression analysis was perfomed with the Affymetrix Gene Arrays HG U133A. Genes with expression level that changed in response to stimuli (termed regulated genes) were selected on the basis of repeated differences in the expression levels of the treated and untreated samples. Of the 22000 genes represented on the oligonucleotide array, in average, a total of 5640 genes changed their expression significantly (fold change > 2.0) upon encounter with one of the pathogen, more than 100 genes showed an increased expression by a factor of > 2.5. Such large-sacle change in gene expression demonstrated that DCs are able to undergo marked transformation in their cellular phenotype. Analysis of the individual responses to pathogens showed that a unique number of genes was regulated by each pathogen. Analysis of Aspergillus hyphae and surprisingly also PpSAB-specific regulated genes showed that DCs strongly and rapidly up-regulated most innate immune genes on the array, including inflammatory cytokines, (e.g. GM-CSF, TN F, IL1 , IL1 , IL6), gro oncogenes and neutrophil and monocyte-attracting chemokines (e.g. MIP3a, RANTES). DC are exquisitely sensitive to different pathogens making them sentinel for innate recognition and initiation of Th cell differentiation.
Full conference title:
Amerian Society of Hematology Annual Meeting
- ASH 44th (2002)